Pseudouridines on Trypanosoma brucei mRNAs are developmentally regulated: Implications to mRNA stability and protein binding

The parasite Trypanosoma brucei cycles between an insect and a mammalian host and is the causative agent of sleeping sickness. Here, we performed high-throughput mapping of pseudouridines (Psi s) on mRNA from two life stages of the parasite. The analysis revealed similar to 273 Psi s, including developmentally regulated Psi s that are guided by homologs of pseudouridine synthases (PUS1, 3, 5, and 7). Mutating the U that undergoes pseudouridylation in the 3' UTR of valyl-tRNA synthetase destabilized the mRNA level. To investigate the mechanism by which Psi affects the stability of this mRNA, proteins that bind to the 3' UTR were identified, including the RNA binding protein RBSR1. The binding of RBSR1 protein to the 3' UTR was stronger when lacking Psi compared to transcripts carrying the modification, suggesting that Psi can inhibit the binding of proteins to their target and thus affect the stability of mRNAs. Consequently, Psi modification on mRNA adds an additional level of regulation to the dominant post-transcriptional control in these parasites.